HMGCS1 Human

What is the function of HMGCS1 in human cells?

HMGCS1 catalyzes the condensation reaction between acetoacetyl-CoA (AcAc-CoA) and acetyl-CoA (Ac-CoA) to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), representing the first committed step in the mevalonate pathway . This cytosolic enzyme is critical for cholesterol biosynthesis and production of isoprenoids essential for protein prenylation, affecting numerous cellular processes including signal transduction and protein trafficking.

To investigate HMGCS1 function experimentally, researchers typically employ several complementary approaches:

• Gene expression modulation using RNA interference or CRISPR-Cas9 technologies to create knockdown or knockout models

• Metabolic labeling with isotopically-tagged precursors to track flux through the mevalonate pathway

• Pharmacological inhibition studies using pathway modulators

• Recombinant protein expression for enzymatic characterization

• Patient-derived cell models harboring HMGCS1 variants

The enzyme's activity influences multiple cellular pathways beyond cholesterol synthesis, including the Hedgehog signaling pathway, where cholesterol directly activates Smoothened (SMO), a G-protein coupled receptor that transmits Hedgehog signals .

Where is HMGCS1 primarily expressed in human tissues?

HMGCS1 demonstrates tissue-specific expression patterns that inform appropriate experimental model selection. RNA-sequencing data from NCBI BioProject and GTEx portal indicate that HMGCS1 is enriched in brain and liver tissues, with lower expression levels in skeletal muscle and other tissues . This differential expression pattern is important for understanding the tissue-specific manifestations of HMGCS1-related disorders.

In myogenic lineages, HMGCS1 expression shows significant variation:

Western blotting confirms that HMGCS1 protein is more abundant in adult human skeletal muscle compared to fetal skeletal muscle . This developmental regulation may have implications for understanding the pathophysiology of HMGCS1-associated rigid spine syndrome, which primarily affects the musculoskeletal system.

To accurately assess tissue-specific expression patterns, researchers should combine multiple complementary techniques:

• Quantitative PCR for sensitive detection of transcript levels

• Western blotting with validated antibodies for protein quantification

• Immunohistochemistry for spatial distribution analysis

• Single-cell RNA sequencing for cell-type specific expression profiling

How does HMGCS1 differ from its mitochondrial paralogue HMGCS2?

HMGCS1 and HMGCS2 are paralogues that catalyze similar biochemical reactions but function in distinct metabolic pathways and cellular compartments. Understanding these differences is essential for designing specific experimental approaches and interpreting research findings.

Despite these functional differences, the enzymes share approximately 67% sequence similarity . Notably, five of the six substituted HMGCS1 residues associated with rigid spine syndrome (Q29, C268, G297, R430, and S447) are conserved in HMGCS2 (Q66, C305, G334, R467, and S484), indicating their functional importance across both paralogues .

For experimental discrimination between the paralogues, researchers can employ:

• Subcellular fractionation to isolate cytosolic versus mitochondrial compartments

• Isoform-specific antibodies for Western blotting and immunoprecipitation

• Gene-specific silencing approaches

• Mass spectrometry with isoform-discriminating peptide analysis

What structural features of HMGCS1 protein are critical for its function?

Investigating the structure-function relationship of HMGCS1 provides insights into how genetic variants affect enzymatic activity and contribute to disease. Several methodological approaches can elucidate these structural features:

• Protein dimerization analysis using size exclusion chromatography has shown that HMGCS1 functions as a dimer, and this quaternary structure is maintained even in disease-associated variants .

• Thermal stability assessment through differential scanning fluorimetry reveals that certain mutations can compromise protein stability. For instance, three of four tested disease-associated mutants exhibited reduced thermal stability compared to wild-type HMGCS1 .

• Secondary structure analysis via circular dichroism spectroscopy can detect subtle conformational changes that may affect catalytic efficiency.

• Enzymatic assays measuring HMG-CoA formation rates can quantify the impact of structural alterations on catalytic function. Studies have shown that some disease-associated variants exhibit subtle changes in enzymatic activity compared to wild-type protein .

These structural investigations provide a mechanistic basis for understanding how specific amino acid substitutions in HMGCS1 lead to pathogenic outcomes. For example, functional studies of recombinant HMGCS1 with disease-associated variants (p.S447P, p.Q29L, p.M70T, p.C268S) demonstrated that while all mutants maintained dimerization, their stability and catalytic properties were differentially affected .

What types of HMGCS1 variants are associated with rigid spine syndrome?

Rigid spine syndrome is characterized by slowly progressive or non-progressive scoliosis, neck and spine contractures, hypotonia, and respiratory insufficiency . Biallelic HMGCS1 variants identified in patients with this condition include both missense and frameshift alterations:

The clinical course can worsen with intercurrent disease or certain drugs in some patients . Muscle biopsies typically reveal irregularities in oxidative enzyme staining with occasional internal nuclei and rimmed vacuoles.

For researchers investigating these variants, several methodological approaches are recommended:

• Comprehensive genetic analysis including segregation studies in families

• In silico prediction tools to assess potential pathogenicity

• Recombinant protein studies to measure effects on stability and activity

• Patient-derived cell models to investigate metabolic consequences

• Correlation between specific variants and clinical phenotypes

It's noteworthy that biallelic hypomorphic variants in downstream enzymes of the mevalonate pathway, including HMGCR and GGPS1, are associated with similar muscular dystrophy phenotypes, suggesting common pathogenic mechanisms .

How do mutations in HMGCS1 affect protein stability and enzymatic activity?

Investigating the functional consequences of HMGCS1 mutations requires a comprehensive biochemical approach. Studies of recombinant HMGCS1 proteins carrying disease-associated variants have revealed differential effects on protein stability and catalytic function.

Thermal stability assessment using differential scanning fluorimetry has shown that three of four tested mutants (p.Q29L, p.M70T, p.C268S) exhibited reduced thermal stability compared to wild-type HMGCS1 . This decreased stability may contribute to protein dysfunction in vivo, potentially through altered protein half-life or improper folding.

Enzymatic activity measurements have detected subtle changes in catalytic function for some variants. Particularly, two of the four tested mutants showed alterations in enzymatic activity compared to wild-type protein . These changes may affect flux through the mevalonate pathway, potentially disrupting cholesterol biosynthesis and other dependent processes.

Protein dimerization analysis by size exclusion chromatography demonstrated that all tested mutants maintained their dimeric state, suggesting that quaternary structure is preserved despite the pathogenic mutations . This indicates that disease mechanisms likely involve aspects of protein function beyond simple structural disruption.

Interestingly, western blot analysis of muscle biopsy from a patient with the p.S447P variant showed comparable HMGCS1 abundance to control samples . This finding suggests that some pathogenic variants may affect function without significantly reducing protein expression or stability in vivo.

A comprehensive experimental approach for investigating HMGCS1 variants should include:

• Biophysical characterization (thermal stability, CD spectroscopy)

• Kinetic enzyme assays (Km, Vmax, catalytic efficiency)

• Cellular studies examining pathway flux and metabolite levels

• Structural analysis to map mutations to functional domains

What experimental models are most suitable for studying HMGCS1 function?

Selecting appropriate experimental models for HMGCS1 research requires careful consideration of the specific research questions, tissue expression patterns, and technical feasibility. A multi-tiered approach often provides the most comprehensive insights.

Cellular Models:

• Hepatocyte cell lines (HepG2, Huh7) are valuable for studying HMGCS1 in cholesterol metabolism given its high expression in liver tissue

• Neuronal cultures can investigate brain-specific functions where HMGCS1 is also highly expressed

• Myoblast/myotube culture systems are particularly relevant for investigating rigid spine syndrome mechanisms

• Patient-derived fibroblasts or induced pluripotent stem cells (iPSCs) offer disease-relevant cellular contexts

Animal Models:

• Mouse models with conditional or tissue-specific Hmgcs1 knockout/knockdown

• CRISPR-engineered mice carrying specific Hmgcs1 mutations that mirror human pathogenic variants

• Zebrafish models for developmental studies and high-throughput screening

Biochemical Systems:

• Purified recombinant HMGCS1 protein for in vitro enzymatic and structural studies

• Cell-free expression systems for protein folding and stability assessments

When comparing in vitro and in vivo models, research has shown that cellular responses can differ significantly between cultured cells and tissue contexts . While similarities may exist at the pathway level, the specific genes altered under these pathways are often different, suggesting that underlying mechanisms of responses differ between isolated cells and complex tissues .

How do in vitro and in vivo models compare when studying HMGCS1 function?

The choice between in vitro and in vivo approaches for HMGCS1 research presents important methodological considerations that can significantly impact experimental outcomes and interpretations.

Transcriptomic analysis comparing in vitro and in vivo responses has revealed that while certain pathway-level similarities may exist, the specific genes altered within these pathways often differ substantially between cells in culture and tissue contexts . This divergence suggests that the underlying mechanisms of cellular responses in isolated cells may not fully recapitulate the complexity of tissue environments.

For HMGCS1 specifically, several factors should guide model selection:

• Pathway Context: The mevalonate pathway involves multiple enzymes and regulatory factors that may be differentially expressed or regulated in various model systems.

• Tissue Interactions: HMGCS1 function in skeletal muscle may depend on interactions with other cell types (neurons, vascular cells) that are absent in monoculture systems.

• Systemic Influences: Hormonal regulation and metabolic feedback mechanisms present in vivo but absent in vitro may significantly affect HMGCS1 function.

• Developmental Dynamics: Studies show HMGCS1 expression varies between adult and fetal muscles , suggesting developmental regulation that may be difficult to model in vitro.

A recommended research strategy involves:

• Beginning with biochemical characterization using recombinant proteins

• Progressing to cellular models with appropriate tissue context

• Validating key findings in animal models that recapitulate human disease features

• Considering "organoid" or "tissue-on-chip" systems as intermediate complexity models

Researchers should carefully select relevant endpoints when substituting animal testing with in vitro approaches , particularly focusing on parameters that are conserved across model systems.

What methodologies are effective for measuring HMGCS1 activity?

Accurate measurement of HMGCS1 enzymatic activity requires specific approaches that distinguish its function from related enzymes and account for the complex regulation of the mevalonate pathway.

Direct Enzymatic Assays:

• Spectrophotometric methods: Monitoring the condensation of acetyl-CoA and acetoacetyl-CoA by measuring the decrease in absorbance at 303 nm as thioester bonds are cleaved

• HPLC-based detection: Quantifying HMG-CoA formation with high sensitivity and specificity

• Radiometric assays: Using 14C-labeled substrates to track product formation

Sample Preparation Considerations:

• Subcellular fractionation: Isolating cytosolic fractions where HMGCS1 is located, avoiding contamination from mitochondrial HMGCS2

• Rapid processing: Minimizing enzyme degradation and maintaining activity

• Buffer optimization: Including appropriate cofactors and stabilizing agents

Functional Assessment in Cellular Systems:

• Metabolic labeling: Using isotope-labeled precursors (13C-acetate) to trace carbon flux through the pathway

• Mass spectrometry: Quantifying mevalonate pathway intermediates and end-products

• Reporter systems: Employing transcriptional reporters responsive to mevalonate pathway activity

When working with tissue samples from patients with HMGCS1 variants, researchers should consider:

• Parallel measurement of protein levels by Western blotting or mass spectrometry

• Correlation of enzymatic activity with clinical severity

• Comparison with other mevalonate pathway enzymes to assess pathway-wide effects

For recombinant protein studies, kinetic analysis should determine Km, Vmax, and catalytic efficiency parameters to quantify the impact of disease-associated mutations. Such approaches have revealed subtle changes in enzymatic activity for certain HMGCS1 variants associated with rigid spine syndrome .

How can recombinant HMGCS1 be used for functional characterization?

Recombinant HMGCS1 protein provides a powerful tool for detailed biochemical characterization, enabling precise analysis of how genetic variants affect protein function. Several methodological approaches have proven effective:

Expression and Purification:

Bacterial expression systems have successfully produced functional human HMGCS1, with appropriate affinity tags facilitating purification while preserving enzymatic activity . Optimizing solubility conditions is critical for obtaining properly folded, active enzyme.

Structural Analysis:

• Size exclusion chromatography has demonstrated that HMGCS1 functions as a dimer, and this quaternary structure is maintained even in disease-associated variants (p.S447P, p.Q29L, p.M70T, p.C268S) .

• Thermal shift assays have revealed differential stability profiles between wild-type and mutant proteins. Three of four disease-associated mutants exhibited reduced thermal stability, providing mechanistic insights into protein dysfunction .

• Circular dichroism spectroscopy enables detection of alterations in secondary structure elements that might contribute to functional deficits.

Enzymatic Characterization:

Enzyme activity assays measuring the condensation of acetyl-CoA and acetoacetyl-CoA have identified subtle functional changes in certain HMGCS1 variants. Two of four tested disease-associated mutants showed alterations in catalytic properties compared to wild-type enzyme .

Mutation Analysis Strategy:

A comprehensive approach to characterizing HMGCS1 variants includes:

• Site-directed mutagenesis to introduce specific mutations

• Parallel purification of wild-type and mutant proteins under identical conditions

• Comparative analysis of biophysical properties (stability, structure)

• Detailed kinetic characterization (Km, Vmax, substrate specificity)

• Correlation of biochemical findings with clinical phenotypes

This biochemical approach has provided valuable insights into how specific amino acid substitutions in HMGCS1 contribute to rigid spine syndrome, demonstrating that disease mechanisms involve alterations in protein stability and subtle changes in enzymatic function rather than complete loss of catalytic activity .

What is the relationship between HMGCS1, cholesterol metabolism, and Hedgehog signaling?

The interconnection between HMGCS1, cholesterol biosynthesis, and Hedgehog (Hh) signaling represents a fascinating convergence of metabolic and developmental pathways with significant research implications.

HMGCS1 catalyzes a critical step in the mevalonate pathway leading to cholesterol production . Beyond its structural role in membranes, research has revealed that cholesterol functions as an instructive signaling molecule in the Hedgehog pathway, a prominent cell-cell communication system in development .

Mechanistically, cholesterol directly activates Smoothened (SMO), an orphan G-protein coupled receptor that transmits the Hedgehog signal across the membrane in all animals . Unlike many GPCRs regulated by cholesterol through their transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD) .

This relationship has several important research implications:

• Dual Roles of Cholesterol: Cholesterol serves both permissive and instructive functions in Hh signaling. While depletion of cholesterol reduces cellular responses to Hh ligands, acute increases in plasma membrane cholesterol are sufficient to activate Hh signaling independently of the ligand .

• Temporal Dynamics: Studies using cyclodextrin-cholesterol complexes (MβCD:cholesterol) to rapidly deliver cholesterol to cells have shown that Hh pathway activation (measured by Gli1 expression) coincides with the loading of cells with cholesterol, starting at approximately 2 hours post-treatment .

• Signaling Mechanism: Cholesterol-mediated activation of the Hh pathway requires SMO activity, as demonstrated by pharmacological inhibition studies and genetic knockout experiments .

For researchers investigating this relationship, several methodological approaches are valuable:

• Pharmacological modulation of HMGCS1 activity to alter cholesterol levels

• Monitoring Hh pathway activation (e.g., Gli1 expression) following manipulation of cholesterol synthesis

• Using fluorescent cholesterol probes to track membrane cholesterol localization

• Employing SMO mutants with altered cholesterol sensitivity to dissect specific mechanisms

How does HMGCS1 expression change during development and differentiation?

HMGCS1 expression exhibits dynamic patterns during development and cellular differentiation that provide important context for understanding its role in health and disease.

Transcriptomic and proteomic analyses of myogenic lineages have revealed significant variation in HMGCS1 expression across different developmental stages and cell types:

These expression patterns have several important research implications:

• The enrichment of HMGCS1 in satellite cells suggests a potential role in muscle stem cell maintenance or function, which may be relevant to understanding muscle disorders associated with HMGCS1 variants .

• The increased expression in adult versus fetal muscle indicates developmental regulation that may reflect changing requirements for cholesterol or isoprenoids during muscle maturation .

• Similar levels of Hmgcs1 detected in mouse fast-twitch (EDL) and slow-twitch (soleus) muscles suggest that fiber-type specificity may not be a major determinant of expression .

For researchers investigating developmental regulation of HMGCS1, several methodological approaches are recommended:

• Temporal expression profiling across developmental stages using qPCR and Western blotting

• In situ hybridization or immunohistochemistry to visualize spatial expression patterns

• Single-cell RNA sequencing to capture cell-type specific expression dynamics

• Reporter constructs driven by the HMGCS1 promoter to track expression in living systems

• Correlation of expression patterns with functional requirements for mevalonate pathway products

What transcriptomic approaches are useful for studying HMGCS1 function?

Transcriptomic methodologies offer powerful approaches for investigating HMGCS1 function in both normal physiology and disease contexts. These techniques can reveal pathway interactions, regulatory mechanisms, and downstream consequences of HMGCS1 perturbation.

Comprehensive Expression Profiling:

• RNA sequencing (RNA-seq) provides genome-wide expression data following HMGCS1 modulation, revealing both direct and indirect effects on gene expression patterns.

• Targeted approaches such as NanoString technology or qPCR arrays can focus on specific pathway components with high sensitivity.

• Single-cell RNA-seq enables identification of cell-specific responses to HMGCS1 alteration, particularly valuable in heterogeneous tissues like muscle.

Pathway Analysis Strategies:

Transcriptomic analysis has revealed that HMGCS1 perturbation affects multiple cellular processes including:

• Core cellular functions (transcription, cell cycle, growth and proliferation)

• Oxidative stress responses

• Fibrosis pathways

• Inflammatory processes

These findings highlight the broad impact of mevalonate pathway disruption on cellular homeostasis.

Comparative Transcriptomics:

When designing transcriptomic experiments, researchers should consider that responses may differ significantly between in vitro and in vivo systems. While similarities may exist at the pathway level, the specific genes altered within these pathways often differ between cell culture and tissue contexts .

This phenomenon has important implications for experimental design:

• Multiple model systems should be employed when possible

• Key findings should be validated across different experimental contexts

• Pathway-level analysis may be more transferable than individual gene changes

Methodological Recommendations:

For optimal transcriptomic investigation of HMGCS1 function:

• Include appropriate time points to capture both immediate and delayed responses

• Compare wild-type and HMGCS1-deficient or variant systems

• Consider parallel profiling of protein and metabolite changes

• Employ bioinformatic approaches that identify enriched pathways and predicted upstream regulators

• Validate key findings using orthogonal techniques (qPCR, Western blotting)

These approaches can identify novel functions, regulatory mechanisms, and potential therapeutic targets related to HMGCS1 biology.

How can HMGCS1 activity be targeted therapeutically in diseases?

Developing therapeutic strategies targeting HMGCS1 requires understanding its role in disease pathogenesis and identifying effective intervention approaches. For HMGCS1-related disorders such as rigid spine syndrome, several therapeutic avenues warrant investigation.

Potential Therapeutic Approaches:

• Enzyme modulation strategies:

  • Small molecule inhibitors or activators that directly target HMGCS1

  • Allosteric modulators that specifically stabilize disease-associated variants

  • Pharmacological chaperones that improve folding and stability of variant proteins

• Genetic approaches:

  • Gene therapy to deliver wild-type HMGCS1 to affected tissues

  • Antisense oligonucleotides to modulate splicing or expression

  • CRISPR-based strategies for correction of specific variants

• Metabolic bypass strategies:

  • Supplementation with downstream products of the mevalonate pathway

  • Modulation of alternative metabolic pathways that can compensate for HMGCS1 dysfunction

  • Dietary interventions affecting cholesterol homeostasis

Clinical Considerations:

The clinical course of rigid spine syndrome associated with HMGCS1 variants can worsen with intercurrent disease or certain medications . This observation suggests that:

• Preventative measures may be an important component of management

• Some drugs may adversely affect pathway function and should be identified

• Personalized medicine approaches based on specific variants may be necessary

Therapeutic Target Validation:

When investigating potential therapeutic approaches, researchers should consider:

• Tissue specificity: While HMGCS1 is widely expressed, disease manifestations primarily affect skeletal muscle, suggesting the need for muscle-targeted interventions .

• Variant-specific effects: Different HMGCS1 variants exhibit varying effects on protein stability and enzymatic activity, potentially requiring tailored therapeutic approaches .

• Pathway interactions: HMGCS1's role in the mevalonate pathway may allow therapeutic targeting of downstream enzymes or products as alternative approaches.

• Biomarker development: Establishing reliable biomarkers of pathway activity and disease progression is essential for clinical trials and treatment monitoring.

Experience with related disorders caused by defects in downstream enzymes like HMGCR and GGPS1, which present with similar muscular dystrophy phenotypes, may inform therapeutic development for HMGCS1-related conditions .

How does HMGCS1 interact with other components of the mevalonate pathway?

HMGCS1 functions within the complex network of the mevalonate pathway, which involves multiple enzymes, regulatory factors, and metabolic intermediates. Understanding these interactions is essential for comprehending pathway regulation and developing targeted interventions.

Pathway Position and Flux Control:

HMGCS1 catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form HMG-CoA, which serves as the substrate for HMG-CoA reductase (HMGCR) . HMGCR catalyzes the rate-limiting step in cholesterol biosynthesis, converting HMG-CoA to mevalonate. The sequential arrangement of these enzymes creates potential for:

• Feedback regulation where end products (cholesterol, isoprenoids) influence upstream enzyme activities

• Substrate competition between parallel pathways

• Metabolic channeling where intermediates are passed directly between pathway components

Coordinated Regulation:

Several regulatory mechanisms ensure balanced flux through the mevalonate pathway:

• Transcriptional control: HMGCS1 expression is regulated by sterol regulatory element-binding proteins (SREBPs) in response to cellular sterol levels

• Post-translational modifications: Phosphorylation, acetylation, and other modifications can rapidly adjust enzyme activity

• Protein-protein interactions: Physical associations between pathway enzymes may form metabolic complexes for enhanced efficiency

Disease Relationships:

Biallelic variants in different mevalonate pathway enzymes produce related but distinct phenotypes:

• HMGCS1 variants cause rigid spine syndrome

• HMGCR variants cause a similar muscular dystrophy

• GGPS1 variants also present with muscular manifestations

This pattern suggests common pathogenic mechanisms throughout the pathway, potentially involving disrupted prenylation of proteins important for muscle function.

Experimental Approaches:

To investigate HMGCS1 interactions within the mevalonate pathway, researchers can employ:

• Metabolic flux analysis using isotope-labeled precursors to track carbon flow through the pathway

• Protein-protein interaction studies (co-immunoprecipitation, proximity labeling) to identify physical associations

• Integrative omics approaches combining transcriptomics, proteomics, and metabolomics to build comprehensive pathway models

• Pharmacological perturbation using inhibitors of specific pathway enzymes to assess compensatory mechanisms

• Genetic interaction screens to identify synthetic lethal or synergistic effects between pathway components